Fate and function of border-associated macrophages in pneumococcal meningitis

• Applicant: Professor Dr. Uwe Ködel
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Clinical Neurology; Neurosurgery and Neuroradiology ; Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 432002731

Pneumococcal meningitis (PM) is one of the most serious infectious diseases of the central nervous system (CNS). Pneumococcal CNS infection generates massive neutrophilic inflammation which causes brain damage and thus contributes to unfavorable disease outcome. PM primarily affects the membranes surrounding the CNS. The CNS boundaries are populated with different subsets of immune cell populations ranging from so-called border-associated macrophages (BAM) over mast cells and dendritic cells to monocytes, neutrophils and lymphocytes, with BAM being the dominant subset under steady-state conditions. Due to their strategic positioning and their diverse repertoires of pattern recognition and opsonic receptors, BAM seem to be central to the orchestration of the inflammatory response to pneumococcal infection. However, current studies on the impact of BAM depletion on meningitis have revealed ambiguous findings, likely due to different study designs and technical issues. Previous work in a zebrafish model of PM and preliminary work of our group suggest that BAM may decline during the disease course, probably due to infection-associated lytic cell death, which may also contribute to the ambiguous findings of previous BAM depletion studies. These (mixed) findings motivate us to characterize the fate and function role of BAM in PM in more detail. The investigations will be done using a well-established mouse model of PM which closely mimics the clinical features of the human disease. For fate-tracking experiments, we will use CX3CR1GFP/+xCCR2RFP/+-transgenic reporter mice, in vivo dye labeling of BAM, post mortem immunostaining using macrophage markers, and combinations of these techniques. The (average) BAM activation state shall be assessed by staining for signature enzymes (namely iNOS and arginase) and real time PCR analysis on FACS-isolated dye-labeled BAM at three characteristic disease stages. To identify BAM cell death, we plan to perform immunhistochemical and/or immunoblot profiling of cell death pathways, focusing on pyroptosis. Next, we intend to evaluate the impact of BAM depletion by intrathecal clodronate liposome application as well as by intrathecal administration of diphtheria toxin (DT) to CD11b-DTR mice on the disease phenotype in early and advanced meningitis. Finally, we want to assess the role of lytic BAM cell death (as well as possible underlying mechanisms) in PM, by using a triple strategy, namely [a] by intrathecal administration of different inhibitors such as the cell death inhibitors VX-765, GSK-782 or NSA, [b] by BAM depletion and reconstitution (using ASC-, GSDMD, and MLKL-deficient macrophages) experiments, and [c] by generating BM chimeric mice with different (e.g., wild type versus ASC-deficient) BAM populations. In our opinion, this research project will markedly improve our knowledge about mechanisms of immunoregulation within the CNS.

• DFG Programme: Research Grants